Droplet-based microfluidic techniques have led to numerous high profile commercial applications in biotechnology, including DNA sequencing, genomic enrichment for sequencing, and digital PCR. Novel biochemical methods are provided herein to overcome significant limitations in these applications. Novel microfluidic methods are provided that are best suited to support the biochemical methods of the invention, and yet also offer substantial improvements for any droplet-based microfluidic application.
In the field, successful implementation of droplet-based techniques has generally required expensive vision systems to orchestrate automated priming and operation of microfluidic circuitry. Tasks shouldered by vision systems typically include filling channels with fluids, measuring droplet sizes during generation, and characterizing the mixing of droplets either with other droplets or with continuous phases. Typically the results are fed back as a part of closed-loop instrument control. Described herein are non-optical methods for performing the same fluidic tasks, greatly reducing the cost and complexity of droplet-based microfluidics.
In brief description, in some aspects the invention is a method for detecting droplets; in other aspects it is a method for generating droplets; in other aspects it is a method for manipulating the contents of droplets; and in yet other aspects it is a method for sorting the contents of droplets. In one embodiment, the invention is a microfluidic circuit comprising four channels meeting at an intersection, such as an ‘X’ shape. Two opposing channels carry continuous phases of aqueous liquid, called the side channels herein, and a third channel—perpendicular to the previous two and called the main channel herein—carries a stream of aqueous droplets into the intersection flowing within a continuous phase of oil. The oil from the main channel phase separates from the aqueous fluids in the side channels producing an oil gap between the aqueous phases in the intersection. The fourth channel is an outlet. Pressure is applied to the side channels, driving their contents into the intersection and contacting individual droplets arriving from the main channel. The fluidic system is poised such that when a droplet arrives from the main channel, it contacts the boluses of fluid emerging from the side channels and merges with them, forming a transient aqueous bridge from one side channel to the other. During this time, some fluid may flow between the droplet and the side channels, changing the contents of the droplet before it is sheared off in flow as it emerges into the outlet channel.
Electrodes are positioned in direct electrical contact with the fluid in the side channels, at least one per channel, and a voltage is applied across the electrodes. The electrical circuit is an open circuit before the arrival of a droplet because the oil-filled intersection is electrically insulating, hence no electrical current flows. On the arrival of a droplet an aqueous bridge forms, closing the electrical circuit allowing current to flow during the excursion of the droplet through the intersection. Once the droplet snaps off after its excursion through the intersection, the oil breaks the electrical circuit again and the current stops until the arrival of the next droplet. In this manner, bursts of electrical current provide a direct observation of droplets that can be used for typical droplet characterizations such as counting droplets and measuring droplet volume.
Additionally, the relative pressures within the droplets and the side channels can be poised allowing flow into or out of the droplets, providing another key feature of a droplet-based fluidic system: the ability to mix reagents. Furthermore, in certain embodiments of the invention the merging of droplets with the continuous phases requires the application of a voltage, such as with surfactant stabilized interfaces. In this method of the invention, the voltage can be pulsed selectively depending on a trigger signature, such as a burst of fluorescence from a sample contained in a droplet, to capture the contents of selected droplets in the side channels or to selectively add reagents to particular droplets. Lastly, the device may be operated as a droplet generator by merging the fluids from the side channels directly without the assistance of a stream of droplets from the main channel. In these methods of the invention, as the boluses of liquid from the side channels make direct contact inside the fluidic intersection, they merge creating a detectable closed electrical circuit before a newly generated droplet snaps off. Thus methods of the invention are provided for generating, mixing, sorting, and measuring droplets, the key aspects of any droplet-based microfluidic system.
Techniques for merging droplets have been disclosed in, for example, U.S. patent application Ser. No. 13/379,782, filed Jun. 25, 2010, entitled “Fluid Injection”, by Weitz et al., published as U.S. Patent Application Publication No. 2012/0132288 on May 31, 2012, that describes a device called the “picoinjector”. The picoinjector consists of a T-junction, or a series of T-junctions, with each T-junction unit consisting of a single continuous phase channel, called the side channel on the stem of the ‘T’, and a main channel across the top of the ‘T’. The pressure of the continuous phase in the picoinjector is poised such that a bolus of liquid extends partially into the fluidic intersection until flow stops from opposition by the Laplace pressure that grows as the bolus protrudes further into the intersection. A stream of droplets in oil flows in the main channel into the intersection, but the droplets do not merge with the protruding continuous phase unless assisted by a high voltage electrical field from nearby electrodes. Once the electrical field is applied, the thin surfactant-stabilized membrane between the amplified droplets and the continuous aqueous phase ruptures allowing flow of the continuous phase into the droplet. After the emerging bolus of combined liquid from the droplet and the flowing continuous phase reaches a critical volume, the bolus snaps off from the continuous phase due to fluidic shearing. As long as the snap occurs before the arrival of the next droplet, the operation succeeds in adding a fixed volume from the continuous phase into each droplet. Also the picoinjector can be run in reverse, extracting the contents of droplets into the side channel for sorting and other applications.
Techniques for merging droplets have also been disclosed in U.S. patent application Ser. No. 13/371,222, filed Feb. 10, 2012, entitled “Methods for Forming Mixed Dropets”, by Yurkovetsky et al., published as U.S. Patent Application Publication No. 2012/0219947 on Aug. 30, 2012, that describes a device called the “lambda-injector” because the stem of the ‘T’ is typically bent away from a right angle. The microfluidic device operates similarly to the picoinjector except the pressure of the continuous phase is increased such that the stable interface between the continuous aqueous phase and the oil is broken. Rather, the continuous phase protrudes far enough into the intersection such that it is repeatedly sheared into discrete droplets, similar to simple droplet generation in a T-junction described by Thorsen et al. (see Phys. Rev. Lett. 86(18), 4163-4166, 2001). However in this method the frequency of droplet generation is set lower than the arrival frequency of a stream of droplets across the top of the ‘T’. Hence the droplets arrive at the intersection before the snap of the continuous phase occurs, and they merge with the emerging bolus of continuous phase under the influence of the high voltage electrical field. The new enlarged bolus snaps off immediately.
Techniques for merging droplets have also been disclosed in International Patent Application. No. PCT/US2012/030811, filed Mar. 28, 2012, entitled “Injection of Multiple Volumes Into or Out of Droplets”, by Abate et al., Published as International Publication No. WO 2012/135259 on Oct. 4, 2012, that describes the use of multiple picoinjectors sequentially or simultaneously to inject multiple controlled volumes into an incoming stream of droplets. While pico- and lambda-injectors have traditionally employed solid electrodes in contact with the fluid intersection, O'Donovan et al. described a different picoinjector that has one electrode in fluidic contact with a side channel and another in fluidic contact with an electrically isolated “faraday mote” (see Lab Chip 12, 4029-4032, 2012).
All of the embodiments of the pico- and lambda-injectors described above have one common trait: disrupting fluidic interfaces by electrical methods is performed by applying a high voltage electrical field through one or more electrically isolated electrodes. In all cases except for one, the electrical field is supplied by a pair of electrodes in close proximity to the merge zone. The one exception, the device from O'Donovan et al. (2012), employs just one proximal fluid electrode, the “faraday mote”. However even in this device it is a high voltage electrical field that is applied to the interface through electrically isolated electrodes. Due to the electrical isolation between the electrodes, none of the aforementioned techniques are capable of carrying a steady and substantial electrical current that reveals droplet dynamics within the device. In contrast, one core of the invention here is that electrical current, not an electrical field, is applied to the interface through external electrodes in fluid contact with the injection channels. Charge accumulation destabilizes the interfaces allowing fluids to merge. Droplet dynamics within the microfluidic device are readily observed and interpreted through perturbations of the electrical current.
Other aspects of the invention include applications of the microfluidic methods described above, or other fluidic methods, for molecular quantitation in general but especially for DNA quantitation. DNA quantitation by polymerase chain reaction (PCR) amplification is an almost universal technique in molecular biology with countless applications. Conventional quantitative PCR (qPCR) is performed by monitoring the reaction product after each thermal cycle during amplification, typically detecting a fluorescence signal that is proportional to the DNA concentration. qPCR measurements are relatively precise within around a factor of two, and when compared against a standard curve they can be accurate to that extent as well (see Baker et al., Nature Methods, 9(6), 541-544, 2012). qPCR reactions can also be multiplexed (quantifying multiple targets within the same reaction mixture) using separate sequence-specific probes that fluoresce at different wavelengths. However qPCR has critical limitations for certain applications. For example, multiplexing qPCR is generally limited to five or fewer reactions due in part to the constrained spectral space but also more importantly to the serious challenge of compatibility between reactions. Hence in practice qPCR is seldom multiplexed. Another important limitation of qPCR is sensitivity for rare genetic variants amidst a large background of wild-type DNA, with sensitivities rarely exceeding 1:100 mutant-to-wild-type ratios (M-Wt) (see Baker et al., above). qPCR is also susceptible to cross-contamination of samples with amplified DNA from previous runs due to the vast amounts of reaction products, called amplicons, generated by amplification.
Digital PCR (dPCR) is a technique that improves on many of the shortcomings of qPCR, and it is based on diluting and dividing a sample into small enough volumes such that each volume is likely to contain either zero or one target DNA molecule, called a limiting dilution (see Sykes et al., Biotechniques, 13(3), 444-449, 1992). In dPCR, amplification reactions are run to the end-point and the number of PCR(+) reactions, indicated by bright fluorescence signals, are compared to the number of PCR(−) reactions, indicated by low fluorescence signals, as a direct measurement of the starting DNA concentration. Using an emulsion format for dPCR, where millions of individual PCR reactions are isolated within neighboring microfluidic droplets approximately 10 pL in volume, extremely high sensitivities exceeding 1:105 M-Wt have been achieved with dPCR (for example, see Pekin et al., Lab Chip, 11, 2156-2166, 2011). Such high sensitivity is enabled by the isolated environment surrounding the mutant molecules: mutant amplification does not compete with the wild-type in dPCR as it otherwise does in bulk reactions with qPCR. Digital PCR also benefits in a different manner from single target encapsulation. Multiplexed reactions are quite simple to develop because the different reactions never compete. At limiting dilution, only one PCR reaction within each droplet actually initiates even though the reaction mixture contains PCR primers for all of the multiplexed reactions. This biochemical simplification enabled Zhong et al. to demonstrate a 9-plex reaction with little more development than a single-plex reaction (Zhong et al., Lab Chip, 11, 2167-2174, 2011). Furthermore, dPCR also allows separate reactions in a multiplexed mixture to be identified by fluorescence intensity as well as color, enabling the discrimination of the 9-plex reaction that would otherwise have been impossible by conventional qPCR considering the spectral limitations of commonly used fluorophores.
Emulsion dPCR is a powerful method for “needle in a haystack” type applications, and it offers significantly greater potential for multiplexing than standard qPCR. However a tradeoff exists between sensitivity and plexity with this approach. Since droplets are discriminated in part by fluorescence intensity, false positive droplets for one reaction that can normally be identified as outliers based on aberrant intensity may erroneously appear as true positive reactions of a different type. For this reason, the number of different targets is often limited to four or fewer while still not achieving the full sensitivity of single-plex reactions.
Emulsion dPCR also presents a carry-over contamination risk. In conventional qPCR carry-over is derisked by sealing the PCR reaction into tubes and plates before thermal cycling, and then the signal is read out afterwards without ever re-opening the container. In contrast, in dPCR the emulsion is typically removed from standard PCR labware after thermal cycling to perform readout on custom instrumentation. Maintaining an unbroken seal on the amplicons requires either custom integration of thermal cycling with serial one-by-one readout of the droplets, or else a mechanical solution is required to contain the amplicons permanently during and after transfer to the reader. Both approaches are costly and awkward.
Aspects of the invention involve solutions to both of the above issues clouding emulsion dPCR. As is described in detail below, the invented method involves encapsulating the nucleic acid sample at limiting dilution in an emulsion and then amplifying the sample. The microfluidic methods of the invention are ideally suited for then entrapping the amplified reaction products (amplicons) within individual hydrogel particles. The DNA from each droplet is sequestered into hydrogel microparticles such that each microgel contains the DNA from one and only one original droplet. In this manner the physical containment of the amplicons shifts from the emulsion format to a solid support. The invented method provides a layer of defense against carry-over contamination by physically immobilizing the amplicons within particles, and it circumvents the limitations on multiplexing in conventional emulsion dPCR by liberating the amplicons from the confined environment of droplets. For example, the amplicons can be washed and probed by hybridization once they are embedded in particles, whereas even this simple procedure is unmanageable for amplicons in an emulsion. With regard to DNA entrapment the invention is not limited to the microfluidic methods of the invention. However the microfluidic methods of the invention are preferred for the biochemical methods of the invention.
One digital PCR technique that is related to the invention is called BEAMing (see Diehl et al., Nature Methods, 3(7), 551-559, 2006). In this approach the target sample is emulsified after bulk pre-amplification into small aqueous droplets ˜10 μm in diameter within an oil carrier solution. The emulsified solution also contains small 1 μm diameter magnetic beads coated with oligonucleotides that are complementary to universal sequences incorporated into the amplicons during pre-amplification. A second round of emulsion PCR extends the bead-bound oligos into entire amplicons. Post-PCR the emulsion is broken, the beads are washed, and then the amplicon-coated beads are probed by sequence-specific hybridization with fluorescent oligonucleotide probes. The beads are read-out using a standard flow cytometer, and the number of fluorescent beads counted corresponds to the initial target concentration.
BEAMing shares a common advantage with the invention: the amplicons are captured on a solid support after emulsion PCR, facilitating downstream analysis with conventional tools for biochemical characterization. However, BEAMing is fundamentally different from the invention in that the droplets in BEAMing contain zero, one, or more beads for solid support. In contrast, in the invention the droplets themselves become the solid support through polymerization, guaranteeing that the contents of each droplet are captured into a particle. Also, in BEAMing bulk pre-amplification is required to incorporate universal tags prior to emulsion PCR, whereas no pre-amplification is required in the invention. These differences translate into numerous important functional advantages for the invention.
In BEAMing, the number of beads per droplet is dictated by a probability distribution. If the droplets are all equally sized, the bead occupancy follows the Poisson distribution. Diehl, et al. (2006) teach that fewer than 20% of the droplets should contain multiple beads, a relatively low bead concentration that results in over 40% of the droplets containing zero beads according to the Poisson distribution. Any template DNA is wasted in droplets without beads, a serious shortcoming for applications requiring high sensitivity. However, it can be difficult to achieve uniform droplet volumes with methods that are also compatible with beads. The highest droplet uniformities that have been reported were achieved by microfluidic techniques, however microfluidic droplet generators are very susceptible to clogging by beads. Instead, emulsification in BEAMing is generally performed by mechanical agitation, resulting in droplets with ˜10-fold variation in diameter, corresponding to ˜1000-fold variation in volume. Such high non-uniformity results in further waste of template if the guideline of 20% maximum occupancy is applied to the largest droplets. This non-uniformity can also pose other challenges related to the wide variation in reaction conditions. For example, PCR may saturate earlier in smaller droplets where the amplicon concentration rises more quickly due to increased confinement. At a limiting dilution, the number of amplicons inside each PCR-positive droplet is always similar during the exponential stage of PCR regardless of droplet size. Consequently the amplicon concentration in the smaller droplets will be larger, resulting in earlier onset of PCR saturation and hence lower overall yield of amplicons. In contrast, droplets in the invention may be very uniformly sized yielding highly uniform reaction conditions. Also, the contents of each droplet in the invention are counted for maximum assay sensitivity.
Pre-amplification is another shortcoming of BEAMing that is avoided by the invention. There are two significant problems with pre-amplification. First, in SNP genotyping assays (an important application for dPCR) the assay must distinguish the difference of a single base pair along a ˜100 bp strand, however during pre-amplification two almost complementary strands from the different alleles can nevertheless still hybridize together resulting in ambiguous or misleading results after emulsion PCR. While thermodynamics favors the perfect match between complementary strands, the specificity of amplicon hybridization is often dictated by non-equilibrium binding kinetics. In other words, the mismatch might not be energetically perfect, but on the time scale of the reaction if the first encounter between two strands is a mismatch, the complex may not dissociate again before entrapment into the emulsion. Furthermore, higher M-Wt ratios increase the probability of mismatches, effectively reducing the number of properly matched mutant amplicons for analysis. Since it is generally standard procedure in BEAMing to disregard beads that contain signal from both alleles, the outcome is reduced sensitivity.
The second issue with pre-amplification is the loss of sensitivity due to polymerase error. DNA polymerases without proof-reading functions mis-incorporate nucleotides at a rate of about 1 in 10,000 base pairs (reviewed by Cha and Thilly, 1993), imposing a fundamental limit on the sensitivity of the assay. In essence, the polymerase creates mutants from the wild-type that are indistinguishable from true mutant-types. Emulsion PCR without pre-amplification overcomes this fundamental limitation by detecting the mixture of wild-type and mutant amplicons that arises from mis-incorporation. Only true mutants yield mutant-only PCR(+) droplets.
In summary, due to the combination of non-uniform emulsions, pre-amplification, and polymerase error, BEAMing is limited to a sensitivity of about 1:10,000 M-Wt, a capability that is 10-100 times lower than achievable by dPCR, the basis of the invention. Furthermore, BEAMing is poorly suited to multiplexing applications because pre-amplification is widely known to introduce unpredictable bias in reaction yield among the different competing reactions. Aspects of the invention avoid all of these shortcomings.
Techniques for encapsulating biomolecules microgels have been disclosed in, for example, International Patent Application No. PCT/CA2005/000627, filed Apr. 25, 2005, entitled “Method of Producing Polymeric Particles with Selected Size, Shape, Morphology and Composition”, by Kumacheva et al., published as U.S. Patent Publication No. 2011/0129941 on Jun. 2, 2011 that describes methods for capturing DNA within hydrogel particles formed from emulsions in which the individual droplets are hardened into particles of pre-determined shapes that are dictated by confinement into microfluidic channels, including spherical shapes. The particles emerge from the microfluidic device rigidified into their final shape. While certain methods of the invention do envision performing some degree of pre-polymerization of droplets within microfluidic channels to stabilize the emulsion, no requirement exists to rigidify the droplets by gelation within a microfluidic device into any particular shape, nor does the invention have a requirement for microfluidic approaches at all. And, while not limited in this regard, spherical particles formed spontaneously by droplets in bulk emulsion are preferred for simplicity in the invented method.
Techniques for confining and synthesizing DNA within microgels have also been disclosed, for example in International Patent Application No. PCT/US08/03185, filed Mar. 7, 2008, entitled “Assays and Other Reactions Involving Droplets”, by Agresti et al., published as U.S. Patent Publication No. 2010/0136544 on Jun. 3, 2010 that describes amplifying individual DNA molecules within gel droplets, fabricated by polymerizing an emulsion containing DNA at limiting dilution, and where one of the PCR primers is covalently incorporated into the gel before amplification. The spatially co-localized clonal amplicons are termed a “polony”, for polymerase colony, and they can be washed, treated, and analyzed similarly to the gel-incorporated amplicons of the invention. However, the similarity in outcome is superficial since the process for fabrication of gel-incorporated clonal amplicons is different for polonies compared to those of the invention. In the method of Agresti et al. (2006) PCR amplification proceeds by extending primers already bound to a pre-formed gel matrix, whereas in the methods of the invention the primers are unbound during amplification and the gel matrix is formed after DNA amplification. One strong advantage of solution-state DNA amplification is PCR efficiency. Diffusion is impeded in the confined environment of pores inside of hydrogels in the method of Agresti et al., with numerous potential pitfalls. First, hindered amplicon diffusion leads to locally high amplicon concentrations, potentially causing early PCR saturation due to amplicon-amplicon interactions competing with primer hybridization and also non-uniform particle loading with amplicons. Second, hindered diffusion of the reverse primer leads to its local depletion, exacerbating the issues above. Third, the forward primer, being bound, cannot diffuse at all. Hence the reaction depends on the diffusion of both the target DNA and the amplicons causing the reaction kinetics to be sluggish especially in the first few replications. Some of the initial template DNA molecules, generally being significantly larger than the amplicons, may also become immobilized during gel polymerization thus preventing any amplification from initiating and resulting in an overall loss in sensitivity. Additionally, the locally high concentrations of DNA and primers caused by confinement increase the opportunity for amplification of non-specific genomic DNA fragments or primer-primer products. Some of these concerns for polonies can be mitigated by increasing the pore size of the particles, however large pore sizes may reduce the efficiency of incorporating amplicons into the gel. Due to such biochemical and other limitations (see Edwards, Jeremy S. “Polony Sequencing: History, Technology, and Applications.” Ed. Michal Janitz. Next-Generation Genome Sequencing: Towards Personalized Medicine. John Wiley & Sons, 2011), the original polony concept (see Mitra and Church, Nucleic Acids Res., 27(24), e34, 1999) actually adopted aspects of BEAMing during its evolution into polony sequencing (see Shendure et al., Science, 309, 1728-1732, 2005). The issues of incorporation efficiency and non-ideal PCR conditions are overcome by methods of the invention.